The long-term objective of this project is to understand better how the activation gate of voltage-gated Na+ channels works during state transitions. As a first step, we plan to delimit the whereabouts of an inner obstruction site critical for activation gating. We hypothesize that the cytoplasmic portions of Na+ channel S6 segments form such a constricted site. Our rationale is based on two S6-situated receptors and their "gated" access for local anesthetics and batrachotoxin. Our specific aims are (1) to create, express, and characterize a series of cysteine-substituted mutants at positions 15-28 of all four homologous S6 segments (D1-S6 to D4-S6), (2) to determine the accessibility of these cysteine-mutants with charged cysteine-modifying reagents, and (3) to create, express, and characterize additional mutants with residues of different size, hydrophobicity, and polarity at this putative constricted site. Mutants of the human heart a-subunit Na+ channel (hH1) clone wifi be expressed in human embryonic kidney cells by transient transfection. Mutant Na+ channels and their gating properties will be first characterized under whole-cell configuration. Cysteine-mutants will be then assessed after internal application of charged cysteine-modifying reagents with and without repetitive pulses to evaluate their "gated" accessibility during state transitions. If needed, in-side-out patches will be used for direct measurements of chemical reactivity rate. Gated and ungated profiles of various cysteine-mutants will allow us to infer the clustered pore-lining residues along the S6 a-helical structures. In addition, UV irradiation of a tethered photo-activatable linker attached to cysteine-mutants may further reveal the S6 movement during channel opening. Subsequent characterizations of the junction between gated- and ungated-accessible region with additional single or double mutations may unravel how such a constricted site opens upon depolarization at the molecular level. This pore-lining site also governs the access of a variety of clinical drugs such as local anesthetics, antiarrhythmics, and anticonvulsants to their receptor(s) within the Na+ channel inner vestibule. Detailed mapping of the cytoplasmic S6 regions along with their linkage with the Na+ channel activation gating may provide insights for the design of new therapeutic drugs that target this important region.